The Journal of Neuroscience, April 1, 2001, 21(7):2240–2246

S100␤ Interaction with Tau Is Promoted by Zinc and Inhibited by Hyperphosphorylation in Alzheimer’s Disease

W. Haung Yu,1,2 and Paul E. Fraser1,3 1Centre for Research in Neurodegenerative Diseases, 2Department of Pharmacology, and 3Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M5S 3H2

The zinc-binding protein S100␤ has been identified as an inter- sistent with an intracellular association. This was enhanced by acting partner with the microtubule-associated protein tau. the addition of zinc and eliminated by divalent metal chelators. Both proteins are individually affected in Alzheimer’s disease S100␤ uptake was also accompanied by extensive neurite (AD). S100␤, is overexpressed in the disease, whereas hyper- outgrowth that may be mediated by its interaction with tau. phosphorylated tau constitutes the primary component of neu- S100␤-tau binding may represent a key pathway for neurite rofibrillary tangles. In this study, we examine factors that mod- development, possibly through S100␤ modulation of tau phos- ulate their binding and the potential role the complex may play phorylation and/or functional stabilization of microtubules and in AD pathogenesis. Zinc was identified as a critical component process formation. S100␤–tau interaction may be disrupted by in the binding process and a primary modulator of S100␤- hyperphosphorylation and/or imbalances in zinc metabolism, associated cellular responses. Abnormally phosphorylated tau and this may contribute to the neurite dystrophy associated extracted from AD tissue displayed a dramatically reduced with AD. capacity to bind S100␤, which was restored by pretreatment with alkaline phosphatase. In differentiated SH-SY5Y cells, ex- Key words: S100␤; tau; Alzheimer’s disease; zinc; binding; ogenous S100␤ was internalized and colocalized with tau con- colocalization; neuronal development

S100␤ is a small molecular weight (10 kDa) zinc–calcium binding This study examines the relationship between tau and S100␤ protein produced by astrocytes (Donato, 1991; Mrak et al., 1995). based on the observation that they are cellular binding partners In addition to metal binding, S100␤ has several functions that and each may therefore regulate specific neurite outgrowth or tau include a role in the cytokine cycle, inhibition of selected phos- hyperphosphorylation activity (Baudier and Cole, 1988; Sorci et phokinases, including phosphokinase C (PKC), and the stimula- al., 2000). Second, tau is a unique neuronal component that tion of neurite outgrowth (Kligman and Marshak, 1985; Baudier stabilizes microtubules leading to the formation of axonal pro- and Cole, 1988; Marshak and Pena 1992; Zimmer et al., 1995; cesses and, in its hyperphosphorylated state, tau is the major Griffin et al., 1998; Heizmann and Cox, 1998). S100␤ is located on component of neurofibrillary tangles (Su et al., 1994; Nagy et al., chromosome 21 and is increased in Down’s syndrome and Alz- 1995; Ikura et al., 1998; Mailliot et al., 1998). Finally, although the heimer’s disease (by as much as 20-fold) (Griffin et al., 1989, 1998; mechanism is unknown, S100␤ can induce a similar neurite Marshak et al., 1992; Castets et al., 1997). In AD, the pathology outgrowth that may be related to its association with tau. S100␤ is defined by amyloid plaques and neurofibrillary tangles (NFT) has been shown to directly affect tau, for example, by its ability to that are accompanied by neuronal loss and aberrant neuritic block PKC phosphorylation at specific sites (Ser 262 and 313) sprouting (Masilah et al., 1991). The neuritic response may be (Biernat et al., 1992; Lin et al., 1994; Singh et al., 1996a). This induced by the loss of neuronal connections or a cellular reaction activity may have a direct consequence for AD because loss of ␤ to amyloid deposition (Mrak et al., 1996). S100 overexpression PKC phosphorylation increases the susceptibility of tau to hyper- in AD has been directly correlated with plaque-associated dys- phosphorylation by GSK-3␤ (Singh et al., 1996b; Tsujo et al., trophic neurite development and the astrocyte activation, as well 2000). This AD-related phosphorylation is considered to be a ␤ as S100 overproduction, may be a direct effect of the loss of major factor in tau deposition and neurofibrillary degeneration ␤ neuronal connections and amyloid- deposition (Van Eldik and (Su et al., 1994; Friedhoff et al., 1998; Ikura et al., 1998; Mailliot ␤ Griffin, 1994; Mrak et al., 1996; Sheng et al., 2000). S100 levels et al., 1998). are elevated in brain regions with a direct relationship to the We have examined S100␤ binding proteins by affinity chroma- presence of neuritic plaques (Sheng et al., 1994). In addition, tography and immunoprecipitation to survey the potential in- ␤ astrocyte activation and S100 expression may also be correlated volvement of other AD-associated proteins. In addition to tau, with neurofibrillary tangle formation in AD (Sheng et al., 1994). S100␤ binding to the amyloid precursor protein (APP), the amyloid-␤ peptide, and the presenilins (PS1 and PS2) were also Received Nov. 15, 2000; revised Jan. 11, 2001; accepted Jan. 18, 2001. assessed. Among the proteins we evaluated, tau was the only This work was supported by the Medical Research Council of Canada, Ontario Mental Health Foundation, and the Alzheimer Society of Ontario. W.H.Y. is significant binding protein and furthermore, based on immuno- supported by an Alzheimer’s Society of Canada Doctoral Award. fluorescence studies, colocalized with S100␤ after internalization Correspondence should be addressed to Haung Yu, Centre for Research in by neuronal cells. Zinc has also been implicated in some aspects Neurodegenerative Diseases, 6 Queen’s Park Crescent West, University of Toronto, Toronto, Ontario, Canada M5S 3H2. E-mail: [email protected]. of AD pathology, such as promotion of amyloid fibril formation Copyright © 2001 Society for Neuroscience 0270-6474/01/212240-07$15.00/0 (Bush et al., 1994) and, when examined in the current system, it Yu and Fraser • S100␤ Binding to Tau J. Neurosci., April 1, 2001, 21(7):2240–2246 2241

significantly affected the relationship between S100␤ and tau. This may be attributable to zinc-induced conformational changes that result in the exposure of a hydrophobic domain and could represent a key site for tau binding (Fujii et al., 1986; Baudier and Cole, 1988; Baudier et al., 1992). In addition, changes to tau also regulated this interaction, as shown by the altered binding of S100␤ to the AD-related hyperphosphorylated NFT-tau. Based on our observations, S100␤-tau binding, overexpression of S100␤, and tau hyperphosphorylation in Alzheimer’s disease pathology suggest that S100␤–tau interactions may contribute to neuronal development as well as neuronal dysfunction. Figure 1. Affinity chromatography using immobilized S100␤ for identi- fication of binding proteins (A). Immunoblotting of zinc (lanes 1, 3, 5, 7)- and EDTA (lanes 2, 4, 6, 8)-eluted fractions indicated a significant amount MATERIALS AND METHODS of S100␤-associated tau in control samples from both frontal (lanes 1, 2) Purification of S100␤. Extracts containing S100␤ were prepared from and temporal cortices (lanes 3, 4). Comparable affinity analysis with fresh bovine brains using the method described by Isobe et al. (1977). A AD-extracted proteins from frontal (lanes 5, 6) or temporal (lanes 7, 8) cortex indicated only weak tau immunoreactivity consistent with a re- 20% homogenate was made in a potassium phosphate buffer (0.1 M duced interaction with S100␤. Zinc-treated samples did not elute any KPO ,pH7.1,1mM EDTA, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 4 proteins with tau immunoreactivity. Immunoblotting of total brain ho- 1mM polymethonyl sulfate) with 2.66 M (or 50%) ammonium sulfate ␤ (AmSO ). Cell debris was removed by centrifugation at 10,000 ϫ g, and mogenates from AD and control indicating the elevated levels of S100 , 4 as has been previously demonstrated by Griffin et al. (1989) (B). the supernatant was adjusted to 85% AmSO4 at pH 4.2 and incubated at 4°C for 2 hr. Precipitated proteins were recovered by centrifugation, dialyzed against phosphate buffer, and stored at Ϫ20°C in lyophilized form. From this crude material, S100␤ was purified using a modified eluted with 500 mM NaCl with 1 mM EDTA. Samples were collected, method as described by Baudier et al. (1982). Crude extracts were dissolved dialyzed, and examined by Western blotting using tau antibodies. ␤ ␤ in the elution buffer (50 mM Tris-Base, pH 7.4) with 1 mM ZnSO and S100 internalization and subcellular distribution. Bovine S100 (final 4 ␮ applied to a Phenyl Sepharose 650 M column (ToyoPearl, Montgomer- concentration, 5 g/ml) was added to culture and incubated for pulse of yville, PA). S100␤ was eluted using a step gradient containing 300 mM 4 or 24 hr. Cells were washed with fresh medium and harvested at 0, 15, NaCl, 0.25 mM ZnSO ,or2mM EDTA. Protein purity was assessed by 30, or 60 min and 4, 24, or 48 hr. Cells lysates were examined by 4 ␤ SDS-PAGE with Coomassie staining and by Western blotting with an immunoblotting to determine cellular uptake of S100 . SH-SY5Y cells S100␤ monoclonal antibody (clone SH-B1; Sigma, St. Louis, MO). were grown in 10% fetal bovine serum/DMEM (Life Technologies, Electrophoresis and Western blotting. S100␤ (1 ␮g) was dissolved in Burlingame, CA) at 37°C under 5% CO2. Cells were placed on poly-L- ␮ Laemmli buffer and separated on a 10–20% Tricine gel (Novex, Carls- lysine-coated coverslips and differentiated using 10 M trans-retinoic ␤ bad, CA). Gels were either stained with 0.2% Coomassie blue reagent in acid. To examine colocalization with tau, S100 was preincubated with ␮ 5% acetic acid, or transferred to a polyvinylidene difluoride membrane. the cells for 4, 12, and 24 hr under control conditions or with 50 M ␮ ␤ ␮ The membrane was washed in Tris-buffered saline (200 mM Tris-base, EDTA or 5 M EGTA for 1 hr before addition of S100 or with 10 g/ml pH 7.4, 150 mM NaCl), blocked with skim milk and incubated overnight ZnSO4. Cells were fixed with 2% paraformaldehyde and examined by with the required antibody. Immunoreactive bands were identified with immunofluorescence using a Nikon TE300 inverted microscope attached HRP-conjugated secondary antibodies and visualized using enhanced to a Bio-Rad Radiance 2000 laser confocal system. chemiluminescence (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL) with film exposure. RESULTS S100␤ affinity chromatography and identification of binding proteins. Identification and analysis of S100␤ binding proteins Purified S100␤ was immobilized on AffiGel-10 (Bio-Rad, Hercules, CA) Interactions of brain-derived proteins, such as tau, were initially and equilibrated in 100 mM HEPES with 0.25 mM ZnSO4. Immobilized S100␤ was incubated with a human brain tissue homogenate (10% w/v), examined by affinity chromatography using immobilized S100␤ as and nonspecific binding proteins were removed by washing with the the primary substrate. A native S100␤ secondary structure was initial buffer. A high salt (100 mM HEPES, 1 M NaCl, 0.25 mM ZnSO4) maintained in the presence of calcium and zinc to obtain physi- wash was used to elute proteins with weak S100␤ interactions. Zinc- ologically relevant conditions for the evaluation of binding pro- dependent binding proteins were subsequently eluted with 1 mM EDTA, and any remaining bound elements were removed with 1 M urea. All teins (Baudier et al., 1982). A series of increasing elution strin- samples were collected and dialyzed then stored at Ϫ20°C in their gencies were used to determine the relative affinities of S100␤ lyophilized form. Eluted proteins were analyzed on 4–20% Tricine gels binding proteins. Proteins that failed to bind to the S100␤ sub- (Novex) and examined by silver staining and by Western blotting. Anti- strate were recovered in the initial wash. This was followed by a bodies corresponding to S100␤, amyloid-␤ (clone 6F/3D; Dako, Carpin- teria, CA), tau (Dako), and a presenilin antisera (Yu et al., 1998) were high salt elution to isolate proteins with weak ionic binding used to determine if they were capable of binding to S100␤. properties. High-affinity S100␤-associated proteins were removed Formation of S100␤ complexes with normal and AD tau. AD and by the addition of zinc chelators, which caused a structural rear- control brain were homogenized (10% w/v) in 0.1 M KH2PO4,2mM rangement of S100␤. Previous studies have shown that zinc ex- EDTA, 2 mM EGTA, and protease inhibitors. Samples were centrifuged for 45 min at 20 000 ϫ g, and the supernatant was fractionated using 35 poses a hydrophobic domain, which represents a potential binding and 55% ammonium sulfate to produce a tau-enriched fraction. Crude site for its cellular partners (Isobe et al., 1977). Finally, any protein precipitates were resuspended in 20 mM Tris and 0.5 M NaCl, pH remaining proteins bound to the affinity column were removed 7.6, with protease inhibitors. Samples were boiled, centrifuged at with a denaturing urea wash, and each of these fractions was ϫ 25,000 g for 30 min, and control aliquots were collected. To assess the examined by direct silver staining as well as Western blotting. effects of phosphorylation on S100␤ binding, samples were also treated with alkaline phosphatase (Sigma) for 30 min at 37°C. Binding of S100␤ Immunoblotting of the various elutions demonstrated that tau with tau from these enriched samples was assessed by immunoprecipita- constituted a principal S100␤ binding protein. All other AD- tion. Aliquots of the brain extracts (50 ␮g of total protein) were com- related proteins such as APP, amyloid-␤, PS1, and PS2 did not ␮ ␤ ␮ ␤ bined with 1 g of purified bovine S100 and 10 l of S100 monoclonal show any significant S100␤ binding and were recovered in the antibody. The mixture was incubated overnight at 4°C and the S100␤- containing complexes were recovered by immunoprecipitation by initial elution. Tau binding was particularly evident in the samples protein-G sepharose. Beads were washed with buffer containing 50 mM obtained from control cases in which strong signals were observed Tris with 150 mM NaCl and 0.5% NP-40, and S100␤ with bound proteins for all brain regions (Fig. 1A). The control tau was only eluted 2242 J. Neurosci., April 1, 2001, 21(7):2240–2246 Yu and Fraser • S100␤ Binding to Tau after zinc chelation with EDTA, suggesting that the observed conformation changes are important for binding. In contrast, in the comparable elutions, there was a marked decrease in the amount from the AD tau fraction (Fig. 1A). The lack of tau was not attributable to loss of immunoreactivity caused by changes in the AD-related protein because a polyclonal, nonphosphorylation- dependent antibody was used. To confirm this, additional antisera were used (e.g., phosphorylation epitopes detected by the antibody AT8), which demonstrated a similar lack of tau binding. Examina- tion of the complete range of elutions revealed that AD-tau was found in both the flowthrough and salt washes. Based on this finding, it was determined that tau from AD samples had a signif- icantly lower affinity for S100␤. To examine potential changes in the S100␤ levels between AD and control cases, Western blotting of comparable tissue samples was investigated. In the AD cases that showed the loss of tau binding to S100␤, appreciable increases in the S100␤ levels were observed in all AD brain samples (Fig. 1B). The reason for the increased expression is unclear but does suggest an imbalance in S100␤ levels that may represent a compensatory mechanism for reduced activity. For example, if S100␤ does modulate tau func- tion and/or metabolism, then the loss of this interaction in AD may induce the elevated expression. Identification of S100␤ and tau complex To assess further the binding of S100␤ to tau, immunoprecipita- tion of in vitro complexes was examined using both AD and control extracted samples. To accomplish this, a tau-enriched fraction was obtained from the brain homogenates through am- Figure 2. Immunoprecipitation of S100␤ complexed with brain-extracted monium sulfate precipitation and incubated with purified S100␤. tau from control and AD cases (3 separate tissue samples). Purified S100␤ The effects of tau phosphorylation on S100␤–tau binding were incubated with tau-enriched and precipitated with an S100␤ polyclonal also examined by immunoprecipitation with untreated extracts as antibody indicated significant interacted evidenced by the coprecipitating tau. Untreated AD extracts displayed reduced tau binding to S100␤ under well as after incubation with alkaline phosphatase. Because AD- comparable conditions. The association was restored by dephosphoryla- tau is heavily phosphorylated, this may be one reason for the tion of the tau-containing extracts using alkaline phosphatase (Alk-Phos.). observed reduction in its binding to S100␤. Immunoprecipitation of untreated AD extracts using an anti- vation of phosphokinases to initiate axonal growth or microtubule S100␤ antibody yielded very low or undetectable levels of associ- stabilization (Baudier et al., 1987a, 1988; Baudier and Cole, 1988; ated tau in all tissues examined (Fig. 2). This finding is consistent Lin et al., 1994; Sheu et al., 1994; Mrak et al., 1996; Sheng et al., with the affinity chromatography results and suggests an impaired 1996). One possibility that we explored was the direct uptake of binding. In contrast, similar immunoprecipitation control samples exogenous S100␤ by neuronal cell lines and the effects of this produced a robust level of binding of tau to S100␤. The high level internalization on tau. Purified S100␤ was added to cultures of of tau immunoreactivity reflects the amount of binding to S100␤ SH-SY5Y cells and was pulsed for 4 or 24 hr and then removed in immunoprecipitation samples relative to the same amount of from the medium. Examination of cell lysates for S100␤ indicated protein used in the AD samples. The formation of the S100␤–tau that after 4 hr of incubation relatively low levels of the S100␤ complex in the control extracts was also zinc-dependent. This dimer were observed (Fig. 3). When examined after different event was demonstrated by the removal of zinc with EDTA, incubation times (4 and 24 hr), the amount of S100␤ slowly followed by the subsequent release of tau from immunoprecipi- decreased with a significant reduction observed at 4 hr and a tated S100␤. This observation is consistent with the elution pro- complete loss of cell-associated protein at 24 hr. Incubation for a file from the affinity column, which facilitated the removal of tau 24 hr period resulted in substantially greater amounts of S100␤ in from the immobilized S100␤. Dephosphorylation of tau by alka- the cell lysates, including both monomeric and dimeric forms line phosphatase restored the normal, possibly functional, bind- (Fig. 3). These levels were maintained 4 hr after incubation and ing of tau to S100␤ (Fig. 2). In all AD cases, we observed a were detectable, but at reduced levels, and they were not observed significantly higher level of binding after tau dephosphorylation. after a 24 hr clearance period. These observations indicate that There was little or no change in the amount of tau that could be significant quantities of S100␤ associate with the cells and are immunoprecipitated in the comparable control samples after al- maintained over long periods of time. kaline phosphatase treatment. Restoration of binding after de- It is unclear from the Western blotting data whether the S100␤ phosphorylation of tau indicates a possible mechanism for the merely accumulates via nonspecific binding to the plasma mem- lack of S100␤–tau interaction in the AD cases. brane or if the cells are capable of internalizing the exogenous Internalization and subcellular distribution of S100␤ in protein. To resolve this issue, retinoic acid differentiated SH- neuronal cells SY5Y cells were used to produce a neuronal-like phenotype and S100␤ has a stimulatory activity on neurite outgrowth that may the distribution of S100␤ examined by immunofluorescence. result from metal influx (calcium), cytokine activation, and acti- Cells incubated with purified bovine S100␤ displayed modest Yu and Fraser • S100␤ Binding to Tau J. Neurosci., April 1, 2001, 21(7):2240–2246 2243

Figure 5. Immunofluorescence of differentiated SH-SY5Y cells demon- strating the effects of zinc on S100␤ internalization. Samples exposed to untreated S100␤ showed an easily detectable level of protein uptake at 24 hr (A). Elevation of the culture medium zinc concentration to 10 ␮M resulted in a substantial increase in the intracellular S100␤ levels (B). This zinc-induced enhancement of S100␤ internalization could be reversed with addition of metal chelators such as EDTA (C). Scale bar, Figure 3. Time course of S100␤ internalization and clearance from 10 ␮m. differentiated SH-SY5Y neuroblastoma cells that were preincubated with S100␤ for 4 or 24 hr. Lysates were examined at different time points (0, 15, 30, and 60 min and 4 and 24 hr) after the removal of S100␤ from the culture medium. Readily detectable S100␤ (monomeric and dimeric other divalent metals) was supported by EDTA treatment that has forms) were observed after the 24 hr pulse and to a lesser extent after 4 a higher affinity for the metal as compared with S100␤. Under hr preincubation. these metal-depleted conditions, the level of S100␤ was markedly reduced in the SH-SY5Y cultures as compared with controls (Fig. 5C). To examine the effects of other divalent cations, the calcium- specific chelator EGTA was added to our cultures to block free and extracellular calcium. Low EGTA concentrations were used because they were not toxic and do not block neuritic sprouting but were sufficient to bind a significant proportion of free calcium. EGTA-treated cells exhibited comparable S100␤ staining, provid- ing additional support for the specific role of zinc (data not shown). Cumulatively, the Western blotting and immunofluores- cence studies suggest that S100␤ is actively internalized by the cells as opposed to surface association. This uptake has a number of implications for the mechanism of S100␤ activity in neuronal systems and its possible relationship to tau function. Colocalization of S100␤ with tau and enhanced neurite outgrowth To investigate the relationship between S100␤–tau binding and neurite outgrowth, differentiated SH-SY5Y cells were allowed to internalize S100␤, and its subcellular distribution with respect to tau was examined by immunofluorescence. Under control condi- tions, S100␤ was broadly distributed within the cell body and some processes. Furthermore, in the double-labeled cells, the staining overlaps to some degree with tau (Fig. 6A). However, a zinc-induced increase in the level of S100␤ within the cell pro- duced a much more defined colocalization with tau. This is Figure 4. Immunofluorescence of SH-SY5Y cells that were preincubated particularly evident within the processes in which the S100␤ and with S100␤ for various lengths of time. Untreated cells displayed very low ␤ ␤ tau coincided as punctate staining that was observed in virtually levels of S100 (A), which were increased after addition of S100 to the ␤ medium and incubations for 4 (B) and 12 hr (C). The S100␤ levels were all neurites (Fig. 6B, arrows). Colocalization of S100 was also significantly increased after 24 hr of incubation (D). S100␤ was distrib- time-dependent because 24 hr of incubation produced higher uted within the cell body and processes consistent with the internalization levels of overlapping signals when compared with the 4 or 12 hr ␮ of the protein rather than cell surface association. Scale bar, 10 m. samples. To ensure that there were no significant changes in tau, the S100␤-treated cells were also analyzed for changes in phos- amounts of intracellular S100␤ staining after exposures for 4 and phorylation using the paired helical filament (PHF)–tau AT8 12 hr (Fig. 4). Consistent with the Western blotting data, sub- antibody. AT8 immunoreactivity was not detected in any of the stantial levels of S100␤ were found after the 24 hr incubation (Fig. treated cells, at any time points (data not shown). The effects of 4D). S100␤ immunoreactivity was distributed within the cell body zinc and the enhanced colocalization may reflect simply an in- and extended into the processes but was absent from the nuclear creased cellular uptake of S100␤, or metal binding may promote region. a preferred conformation that facilitates tau binding. This latter The degree of S100␤ internalization was also affected by zinc, possibility would be consistent with our affinity chromatography as shown by the increased level of staining within cells, as com- and immunoprecipitation results. These findings suggest that in- pared with control, when zinc was added to the medium and ternalized S100␤ may be associated with tau and thereby affect coincubated for 24 hr (Fig. 5). The effect of zinc (and possibly tau function and/or metabolic events such as phosphorylation. 2244 J. Neurosci., April 1, 2001, 21(7):2240–2246 Yu and Fraser • S100␤ Binding to Tau

pathways that are regulated by the two proteins. For example, S100␤ may provide a scaffolding structure for tau to stabilize microtubules and possibly contribute to the abnormal neuritic dystrophy that is observed in AD (Baudier and Cole, 1988; Tam, 1990; Azmitia et al., 1995). This is illustrated by our observation that nonphysiological sprouting of processes are from the cell body, which is not normally seen in differentiated neuronal cul- tures. The second possibility is that S100␤ is a modulator of tau phosphorylation and that any changes in their interaction could Figure 6. Colocalization of internalized S100␤ with tau in differentiated be a factor in the AD-related hyperphosphorylation, as has been neuroblastoma cells. Under control conditions, S100␤ (red) that was taken previously suggested (Baudier et al., 1987a; Sorci et al., 2000). up by the cells showed partial overlap with tau ( green), suggesting a Furthermore, the ability of S100␤ to inhibit PKC may potentiate possible intracellular association (A). The colocalization was more pro- the aberrant phosphorylation at key sites [e.g., residues 262 and nounced with the addition of zinc to the culture medium (B). Zinc elevated levels of S100␤ resulted in increased neurite outgrowth and 313 (Correas et al., 1992; Singh et al., 1996b)]. However, in our in frequent overlap of S100␤ with tau in these processes, which appear as vitro studies, S100␤ did not appear to promote aberrant phos- discrete, punctate staining within the cell processes (B). Addition of phorylation, as indicated by the lack of AT8 staining that identi- ␤ EDTA to the culture medium before incubation of the cells with S100 fies PHF–tau related phosphorylated epitopes (Biernat et al., eliminated the tau colocalization pattern caused by reduced protein uptake (C). Scale bar, 10 ␮m. 1992). Neuritic development, although beneficial in the short term to rejuvenate lost neuronal connections, can also be detri- mental in the chronic stages of AD because it increases cellular metabolic requirements and exposes the neurons to external insults. Initially, our finding that S100␤ failed to bind AD-derived tau was attributed to the reduced number of neurons, which is asso- ciated with the progression of AD. This did not appear to be the case because the normal binding could be restored after alkaline phosphatase treatments. Although this may suggest that all phos- Figure 7. Stimulation of neurite outgrowth in SH-SY5Y cells after phate groups on tau hinder S100␤ binding, this is not evident ␤ S100 internalization. Retinoic acid differentiated cells displayed a because tau is naturally phosphorylated, and this does not affect neuron-like morphology but with only a limited number of extensions (A, ␤ arrow). With the addition of untreated S100␤, the number and length of binding of the control sample tau to S100 . In these studies, it is the processes were enhanced (B). Addition of zinc to the medium and the only with abnormal hyperphosphorylation of tau present in AD accompanying increase in S100␤ uptake resulted in widespread increase that prevents S100␤–tau binding activity. Our study has also in neurite outgrowth, leading to the formation of dense networks of cell demonstrated that zinc is important factor in the internalization C processes ( ). Cells and processes were visualized by immunofluores- of S100␤ into neurons and enhances tau binding. In addition, we cence staining of the cell surface cadherins. Scale bar, 10 ␮m. observed an increase in neuritic sprouting in SH-SY5Y cells treated with S100␤ and zinc, which suggests that metal binding Tau is one of the key elements that control axonal growth may be critical to this outgrowth activity. ␤ and may be modulated, to some degree, by interactions with S100 has been demonstrated to have several biological func- S100␤. This hypothesis is supported in our experimental sys- tions in AD. This is reflected by its ability to bind zinc and tem by the response of the SH-SY5Y cells to S100␤ and zinc. calcium, as well as inhibit certain phosphorylation pathways. In ␤ Even with retinoic acid differentiation, SH-SY5Y cells do not addition, S100 has been shown to activate the complement produce extensive process formation and have a predomi- pathway through interleukin-6 activation (Stanley et al., 1994; ␤ nantly “spindle-type” morphology (Fig. 7A). With the addition Mrak et al., 1995; Sheng et al., 1996a,b; Hays, 1998). S100 itself of S100␤, a greater number of neurites were observed when is activated by interleukin-1 and may also participate in a positive visualized using an antibody staining for cadherins on the cell feedback loop, thereby inducing its own production through the surface (Fig. 7B). Neurite outgrowth was even more pro- promotion of astrocytic activity (Mrak et al., 1995). In AD, the ␤ nounced in the presence of zinc in which enhanced S100␤ observed increase in S100 production appears to be related to uptake resulted in increased number of neurites with extensive some of the physiological changes associated with interleukins ␤ outgrowth that produced both longer networks of processes and to the increase in neuritic sprouting. The uptake of S100 (Fig. 7C). Under these conditions, abnormal neuritic sprouting may represent a key role in its ability to alter the neuronal ␤ was also observed with processes emanating from the cell body. activity. Our immunofluorescence data suggests that S100 up- Stimulation of neurites and colocalization of S100␤ with tau take by cells is enhanced by the addition of zinc. As stated ␤ provides additional evidence for a physiological role for their previously, zinc causes S100 to undergo a conformational interaction. change, exposing a hydrophobic domain that facilitates neuronal internalization. Within the cell, S100␤ may alter many cellular DISCUSSION processes, including binding to tau. These studies were performed to establish the binding of S100␤ The metal-binding capacity of S100␤ appears to be a crucial to tau and the chemical properties involved, as well as identify its functional element and may have some bearing on other disease relevance to Alzheimer’s disease. Our findings demonstrate that pathways. S100␤–calcium effects have been extensively examined S100␤ binds to tau. In addition, this interaction is enhanced by by Baudier and Cole (1987a,b, 1988), in which they found evi- zinc and inhibited by tau hyperphosphorylation. The functional dence of S100␤–calcium binding to microtubule-associated pro- aspects of S100␤–tau binding may impact on several different teins, including tau, and calcium–calmodulin-dependent protein Yu and Fraser • S100␤ Binding to Tau J. Neurosci., April 1, 2001, 21(7):2240–2246 2245 kinase II. Calcium is also thought to be excitotoxic in AD (Kim associated protein tau monitored by fluorescence in solution. Biochem- istry 37:10223–10230. et al., 2000). In AD, both calcium and zinc have been implicated Fujii T, Gochou N, Akabane Y, Fujii M, Kondo Y, Suzuki T, Ohki K in the amyloid toxicity pathway. Zinc, as well as copper, is (1986) Effect of zinc ions on the interaction of S-100 protein with brain believed to accelerate the formation of amyloid fibrils (Bush et al., microtubule. Chem Pharm Bull 34:5040–5044. Griffin WS, Stanley LC, Ling C, White L, MacLeod V, Perrot LJ, White 1994; Yang et al., 2000). Amyloid is implicated as a potential III CL, Araoz C (1989) Brain interleukin 1 and S-100 immunoreactiv- membrane protein that may promote the influx of calcium across ity are elevated in Down syndrome and Alzheimer disease. Proc Natl the plasma membrane. The increase of S100␤ in AD may con- Acad Sci USA 86:7611–7615. Griffin WS, Sheng JG, McKenzie JE, Royston MC, Gentleman SM, tribute to the shuttle of these metals to points of interaction, Brumback RA, Cork LC, Del Bigio MR, Roberts GW, Mrak RE thereby accelerating the pathogenic process. (1998) Life-long overexpression of S100beta in Down’s syndrome: im- plications for Alzheimer pathogenesis. Neurobiol Aging 19:401–405. Zinc does not normally appear in the cell as a free, or unbound, Hays SJ (1998) Therapeutic approaches to the treatment of neuroin- form. It is believed to be toxic in this state. This may be related flammatory diseases. Curr Pharm Des 4:335–48. to the ability of free zinc to enter via AMPA channels (Sensi et Heizmann CW, Cox JA (1998) New perspectives on S100 proteins: a multifunctional Ca(2ϩ)-, Zn(2ϩ)- and Cu(2ϩ)-binding protein family. al., 1997, 1999; Yin et al., 1998), promoting excitotoxicity. Pro- Biometals 11:383–397. teins such as metallothionein and S100␤ are induced by astrocytes Ikura Y, Kudo T, Tanaka T, Tanii H, Grundke-Iqbal I, Iqbal K, Takeda to compensate for the extrusion of zinc into the extracellular M (1998) Levels of tau phosphorylation at different sites in Alzheimer ␤ disease brain. NeuroReport 9:2375–2379. space to block its toxic effects. In the case of S100 , the effect may Isobe T, Nakajima T, Okuyama T (1977) Reinvestigation of extremely detrimentally alter the disease process. acidic proteins in bovine brain. Biochim Biophys Acta 494:222–232. Kim HS, Park CH, Cha SH, Lee JH, Lee S, Kim Y, Rah JC, Jeong SJ, The role of zinc in AD has generated several interesting and Suh YH (2000) Carboxyl-terminal fragment of Alzheimer’s APP de- pathogenically significant hypotheses. The potential role that it stabilizes calcium homeostasis and renders neuronal cells vulnerable to may play with S100␤ on the effect on neuritic sprouting is another excitotoxicity. FASEB J 14:1508–1517. Kligman D, Marshak DR (1985) Purification and characterization of a important addition to this metals role in the disease process. neurite extension factor from bovine brain. Proc Natl Acad Sci USA Finally, our observations suggest that, in addition to its activation 82:7136–7139. of cytokines, S100␤ may also play a more direct role in tau- Lin LH, Van Eldik LJ, Osheroff N, Norden JJ (1994) Inhibition of protein kinase C and casein kinase II-mediated phosphorylation of related pathways that are associated with neurodegeneration in GAP-43 by S100 beta. Brain Res Mol Brain Res 25:297–304. Alzheimer’s disease. Mailliot C, Sergeant N, Bussiere T, Caillet-Boudin ML, Delacourte A, Buee L (1998) Phosphorylation of specific sets of tau isoforms reflects REFERENCES different neurofibrillary degeneration processes. FEBS Lett 433:101–104. Azmitia EC, Rubinstein VJ, Strafaci JA, Rios JC, Whitaker-Azmitia PM Marshak DR, Pena LA (1992) Potential role of S100 beta in Alzheimer’s (1995) 5-HT1A agonist and dexamethasone reversal of para- disease: an hypothesis involving mitotic protein kinases. Prog Clin Biol chloroamphetamine induced loss of MAP-2 and synaptophysin immu- Res 379:289–307. noreactivity in adult rat brain. Brain Res 677:181–192. Marshak DR, Pesce SA, Stanley LC, Griffin WS (1992) Increased S100 Baudier J, Cole RD (1987a) Reinvestigation of the sulfhydryl reactivity beta neurotrophic activity in Alzheimer’s disease temporal lobe. Neu-

in bovine brain S100b (betaϩ beta) protein and the microtubule- robiol Aging 13:1–7. associated tau proteins. Ca 2 stimulates disulfide cross-linking be- Masilah E, Mallory M, Hansen L, Alford M, Albright T, DeTeresa R, tween the S100b beta-subunit and the microtubule-associated tau(2) Terry R, Baudier J, Saitoh T (1991) Patterns of aberrant sprouting in protein. Biochemistry 27:2728–2736. Alzheimer’s disease. Neuron 6:729–739. Baudier J, Cole RD (1987b) Phosphorylation of tau proteins to a state Mrak RE, Sheng JG, Griffin WS (1995) Glial cytokines in Alzheimer’s like that in Alzheimer’s brain is catalyzed by a calcium/calmodulin- disease: review and pathogenic implications. Hum Pathol 26:816–823. dependent kinase and modulated by phospholipids. J Biol Chem Mrak RE, Sheng JG, Griffin WS (1996) Correlation of astrocytic S100 262:17577–17583. beta expression with dystrophic neurites in amyloid plaques of Alzhei- Baudier J, Cole RD (1988) Interactions between the microtubule- mer’s disease. J Neuropathol Exp Neurol 55:273–279.

associatedϩ tau proteins and S100b regulate tau phosphorylation by the Nagy Z, Esiri MM, Jobst KA, Morris JH, King EM, McDonald B, Ca2 /calmodulin-dependent protein kinase II. J Biol Chem Litchfield S, Smith A, Barnetson L, Smith AD (1995) Relative roles of 263:5876–5883. plaques and tangles in the dementia of Alzheimer’s disease: correla-

Baudier J, Briving C, Deinum J, Kaglid K, Sorskogϩ L, Wallin M (1982) tions using three sets of neuropathological criteria. Dementia 6:21–31. Effect of S100 proteins and calmodulin on Ca 2 -induced disassembly Sensi SL, Canzoniero LM, Yu SP, Ying HS, Koh JY, Kerchner GA, Choi of brain microtubule proteins in vitro. FEBS Lett 148:231–234. DW (1997) Measurement of intracellular free zinc in living cortical Baudier J, Mochly-Rosen D, Newton A, Lee SH, Koshland Jr DE, Cole neurons: route of entry. J Neurosci 17:9554–9564. 2ϩ RD (1987) Comparison of S100b protein with calmodulin: interac- Sensi SL, Yin HZ, Weissϩ H (1999) Glutamate triggers preferential Zn tions with mellitin and microtubule-associated tau proteins and inhibi- flux through Ca 2 permeable AMPA channels and consequent ROS tion of phosphorylation of tau proteins by protein kinase C. Biochem- production. NeuroReport 10:1723–1727. istry 26:2886–2893. Sheng JG, Mrak RE, Griffin WS (1994) S100 beta protein expression in Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ (1992) Alzheimer disease potential role in the pathogenesis of neuritic Characterization of the tumor suppressor protein p53 as a protein plaques. J Neurosci Res 39:398–404. kinase C substrate and a S100b-binding protein. Proc Natl Acad Sci Sheng JG, Ito K, Skinner RD, Mrak RE, Rovnaghi CR, Van Eldik LJ, USA 89:1627–1631. Griffin WS (1996a) In vivo and in vitro evidence supporting a role for Biernat J, Mandelkow EM, Schroter C, Lichtenberg-Kraag B, Steiner B, the inflammatory cytokine interleukin-1 as a driving force in Alzhei- Berling B, Meyer H, Mercken M, Vandermeen A, Goedert M (1992) mer pathogenesis. Neurobiol Aging 17:761–766. The switch of tau protein to an Alzheimer-like state includes the Sheng JG, Mrak RE, Rovnaghi CR, Kozlowska E, Van Eldik LJ, Griffin phosphorylation of two serine-proline motifs upstream of the microtu- WS (1996b) Human brain S100 beta and S100 beta mRNA expression bule binding region. EMBO J 11:1593–1597. increases with age: pathogenic implications for Alzheimer’s disease. Bush AI, Pettingell WH, Multhaup G, Paradis MD, Vonsattel J-P, Neurobiol Aging 17:359–363. Gusella JF, Beyreuther K, Masters CL, Tanzi RE (1994) Rapid in- Sheng JG, Mrak RE, Bales KR, Cordell B, Paul SM, Jones RA, Wood- duction of Alzheimer A␤ amyloid formation by zinc. Science ward S, Zhou XQ, McGinness JM, Griffin WS (2000) Overexpression 265:1464–1467. of the neuritotrophic cytokine S100beta precedes the appearance of Castets F, Griffin WS, Marks A, Van Eldik LJ (1997) Transcriptional neuritic beta amyloid plaques in APPV717F mice. J Neurochem regulation of the human S100 beta gene. Brain Res Mol Brain Res 74:295–301. 46:208–216. Sheu FS, Azmitia EC, Marshak DR, Parker PJ, Routtenberg A (1994) Correas I, Diaz-Nido J, Avila J (1992) Microtubule-associated protein Glial-derived S100b protein selectively inhibits recombinant beta pro- tau is phosphorylated by protein kinase C on its tubulin binding tein kinase C (PKC) phosphorylation of neuron-specific protein F1/ domain. J Biol Chem 267:15721–15728. GAP43. Brain Res Mol Brain Res 21:62–66. Donato R (1991) Perspectives in S100 protein biology. Cell Calcium Singh TJ, Wang JZ, Novak M, Kontzekova E, Grundke-Iqbal I, Iqbal K 12:713–726. (1996a) Calcium/calmodulin-dependent protein kinase II phosphory- Friedhoff P, Schneider A, Mandelkow EM, Mandelkow E (1998) Rapid lates tau at Ser-262 but only partially inhibits its binding to microtu- assembly of Alzheimer-like paired helical filaments from microtubule- bules. FEBS Lett 387:145–148. 2246 J. Neurosci., April 1, 2001, 21(7):2240–2246 Yu and Fraser • S100␤ Binding to Tau

Singh TJ, Zaidi T, Grundke-Iqbal I, Iqbal K (1996b) Non-proline depen- kinase-3B by protein kinase C activation: implications for regulation of dent protein kinases phosphorylate several sites found in tau from tau phosphorylation. FEBS Lett 469:111–117. Alzheimer disease brain. Mol Cell Biochem 154:143–151. Van Eldik LJ, Griffin WS (1994) S100 beta expression in Alzheimer’s Sorci G, Agneletti AL, Donato R (2000) Effects of S100 and S100B on disease: relation to neuropathology in brain regions. Biochim Biophys microtubule stability. An in vitro study using triton-cytoskeletons from Acta 1223:398–403. astrocyte and myoblast cell lines. Neuroscience 99:773–783. Yang DS, McLaurin J, Qin K, Westaway D, Fraser PE (2000) Examin- Stanley LC, Mrak RE, Woody RC, Perrot LJ, Zhang S, Marshak DR, ing the zinc binding site of amyloid-␤ peptide. Eur J Biochem 267:1–7. Nelson SJ, Griffin WS (1994) Glial cytokines as neuropathogenic fac- Yin HZ, Ha DH, Carriedo SG, Weiss JH (1998) Kainate-stimulated tors in HIV infection: pathogenic similarities to Alzheimer’s disease. 2ϩ 2ϩ J Neuropathol Exp Neurol 53:231–238. Zn uptake labels cortical neurons with Ca -permeable AMPA/ Su JH, Cummings BJ, Cotman CW (1994) Early phosphorylation of tau kainate channels. Brain Res 781:45–55. in Alzheimer’s disease occurs at Ser-202 and is preferentially located Yu G, Chen F, Levesque G, Nishimura M, Zhang DM, Levesque L, within neurites. NeuroReport 5:2358–2362. Rogaeva E, Xu D, Liang Y, Duthie M, St. George-Hyslop PH, Fraser Tam PK (1990) An immunohistological study of the human enteric ner- PE (1998) The presenilin 1 protein is a component of a high molecular vous system with microtubule-associated proteins. Gastroenterology weight intracellular complex that contains beta-catenin. J Biol Chem 99:1841–1844. 273:16470–16475. Tsujo I, Tanaka I, Kudo T, Nishikawa T, Shinozaki K, Grunde-Iqbal I, Zimmer DB, Cornwall H, Landar A, Song W (1995) The S100 protein Iqbal K, Takeda M (2000) Inactivation of glycogen synthetase family: history, function, and expression. Brain Res Bull 37:417–429.